1. Field of the Invention
The present invention relates to an analysis element for measuring glycated hemoglobin (HbA.sub.1, also referred to as hemoglobin A.sub.1) content ratio, by which amount of glycated hemoglobin and total hemoglobin are simultaneously and separately analyzed and the glycated hemoglobin ratio is calculated. The present invention also relates to an analysis method of glycated hemoglobin content ratio using this element. Particularly, the present invention relates to an analytical element for measuring the glycosylation level of hemoglobin by analyzing the amount of hemoglobin A.sub.1c, which is one of classes of glycated hemoglobins, and total hemoglobin simultaneously and the glycosylation level of hemoglobin is calculated, and relates to the analysis method of the glycosylation level of hemoglobin using this element.
2. Description of the Related Art
Hemoglobin (Hb) is a respiratory pigment present in erythrocyte, which is largely responsible for oxygen transport. A hemoglobin molecule comprises four polypeptide subunits (respectively two .alpha. chain systems and .beta. chain systems), each subunit is formed by association of one globin protein and one heme molecule which is an iron-protoporphyrin complex. Major class of hemoglobin (more than 90%) found in normal adult hemolysate is adult hemoglobin (HbA: also referred to HbA.sub.0 for distinguishing from glycated hemoglobin HbA.sub.1 described hereinafter) having .alpha..sub.2 .beta..sub.2 subunits composition; and trace components such as HbA.sub.2 (.alpha..sub.2 .delta..sub.2) are also found in normal adult.
Among classes of adult hemoglobin HbAs, there is known a glycated hemoglobin (HbA1, also referred to as glycosylated hemoglobin), which may be further classified fractionated into HbA.sub.1a1, HbA.sub.1a2, HbA.sub.1b, and HbA.sub.1c with an ion exchange resin fractionation. All of these subclasses have the same primary structure, which is stabilized by formation of an aldimine (Schiff base) by the amino group of N-terminal valine in .beta. subunit chain of normal hemoglobin HbA and glucose (or, glucose-6-phosphate or fructose) followed by formation of ketoamine by Amadori rearrangement.
In particular, glycated hemoglobin bound with glucose is called HbA.sub.1c (glycosylated hemoglobin, hereinafter also referred to as hemoglobin A.sub.1c), and comprises main portion of glycated hemoglobins. The content ratio of glycosylated hemoglobin is proportional to blood glucose level, and ranges 3-6% in total hemoglobin Hb of normal human adult, though it can go up to 15% in that of diabetics. Hence, determination of the amount of glycosylated hemoglobin HbA.sub.1c is considered to be a good index for carbohydrate metabolism control. In addition, since ketoamine formed by a non-enzymatic reaction with blood glucose is stable, this glycosylated hemoglobin HbA.sub.1c is never decomposed during the life of erythrocyte (120 days in average). Thus, the amount of hemoglobin A.sub.1c in blood is understood to memorize blood glucose levels of last 1 or 2 months. Accordingly, blood glucose levels of the last two months or so can be estimated on the basis of the ratio of HbA.sub.1c to total hemoglobin Hb. Analysis of the hemoglobin A.sub.1c in blood is used as a measurement enabling long-term control of blood glucose level, whereas blood glucose level which goes up for a temporary short period after eating is used as an index of a short-term blood glucose level.
Although a hemoglobin A.sub.1c analysis has been conventionally performed by column chromatography using HPLC (high pressurized liquid chromatography) or mini columns, the column method not only requires an expensive measuring apparatus for exclusive use, but also takes time for the analysis, and thus was not suitable for clinical examinations in which a number of specimens are to be handled.
In recent years, HbA.sub.1c analyses by a turbidimetric immunoassay method or ELISA (enzyme labelled immunosorbent assay) have been proposed (e.g. Unexamined Japanese Patent Publication Nos. 277967/1988 and 46566/1991). However, measurement operation of prior art is complicated, and the analysis requires skill. In addition, there is inconvenient in that the total hemoglobin Hb amount has to be measured by another method although the HbA.sub.1c content ratio is possible to be analyzed by the above mentioned method. Similar problems are also raised in the analyses of glycated hemoglobins other than HbA.sub.1c. Hence, there is a demand for more simple and rapid method for analyzing the ratio of HbA.sub.1c or other glycated hemoglobin to total hemoglobin with high sensitivity.